Compositions for treating amyotrophic lateral sclerosis

ABSTRACT

The present invention relates to compositions and methods for the treatment of amyotrophic lateral sclerosis. More specifically, the present invention relates to novel combinatorial therapies for treating amyotrophic lateral sclerosis or a related disorder.

FIELD OF THE INVENTION

The present invention relates to compositions and methods for treatingamyotrophic lateral sclerosis and related disorders. More specifically,the present invention relates to novel combinatorial therapies for thetreatment of amyotrophic lateral sclerosis and related disorders.

BACKGROUND OF THE INVENTION

Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease,is the most frequent adult motor neuron disease. It was first describedin 1869 by the French neurologist Jean-Martin Charcot. This disease ischaracterized by degeneration and death of motor neurons, which leads togeneralized weakness and muscle atrophy. The course of the disorder isinexorably progressive, with 50% of patients dying within 3 years ofonset. ALS appears as a rare disease with a prevalence of 4-6 per 100000 each year and an incidence of 1-2 per 100 000 each year.

Most cases (90%) are classified as sporadic ALS (SALS), and theremainder 10% are inherited and referred to as familial ALS (FALS), witha Mendelian pattern of inheritance. From a clinical standpoint, familial(FALS) and sporadic (SALS) cases cannot be distinguished from oneanother, apart from a mean age at onset for SALS that is 10 years laterthan for FALS (56 years versus 46 years) [1]. The causes for most casesof ALS are unknown and the clinical course is highly variable,suggesting that multiple factors underlie the disease mechanism. Fewtreatments are available.

The hallmark of this disease is the selective death of motor neuronslocated in the brainstem, motor cortex and spinal cord leading toparalysis of voluntary muscles. The paralysis begins focally anddisseminates in a pattern that suggests that degeneration is spreadingamong contiguous pools of motor neurons. Mortality normally results whencontrol of the diaphragm is impaired and the ability to breathe is lost.

ALS is characterized by progressive manifestations of dysfunction ofboth lower and upper motor neurons. Lower motor neurons connect thebrainstem and spinal cord to muscle fibres. Their dysfunction leads tomuscle atrophy, cramps and fasciculations (small, local, involuntarymuscle contraction). Upper motor neurons originate in motor region ofthe cerebral cortex or the brainstem and carry motor information down tomotor neurons that are directly responsible for stimulating the targetmuscle. Their dysfunction leads to spasticity (continuous musclecontraction that interfere with gait, movement, and speech) andpathological reflexes [2]. The other related motor neuron diseases areusually distinguished by the type of nerve cells impaired, i.e. upper orlower motor neurons: they are known as primary lateral sclerosis (PLS),progressive muscular atrophy (PMA), pseudobulbar palsy and progressivebulbar palsy (PBP).

Diagnosis of ALS is based on clinical signs, established by neurologiston the basis of history, topographic distribution of the neuronal lossand the finding of some characteristic cytological changes. However,there is no clear-cut diagnostic test of ALS available. Clinicalfeatures are classified in accordance with affected neurological regionsthat are bulbar, cervical, and lumbar.

As already mentioned, degeneration in ALS predominantly affects themotor system. However, cognitive and behavioral, as well as sensorysymptoms have recently been reported [3,4] and there is evidence foroverlap between frontotemporal dementia (FTD) and ALS both clinicallyand pathologically [5].

Some weak evidences suggest that ALS onset can be triggered by putativeenvironmental factors [6,7].

Several mutated genes or genomic regions have been reported to cause orpredispose to ALS as well as to ALS with FTD [8-10]. For instance, about20-25% of all FALS cases and around 1% of SALS cases arise because ofmutations in superoxide dismutase SOD1 [11]. Multiple clinicalpresentations within the same family are obtained with the same SOD1mutation which does not necessarily cause a homogenous phenotype. Thereis no clear correlation between enzyme activity, clinical progressionand disease phenotype. However, the period of the disease is similarwhatever the mutation is. Historically, the discovery of SOD1 mutationsled to the generation of the first animal models of ALS. They develop amotor neuron disease closely resembling human ALS [12,13]. Among othergenes implicated in development of ALS or related motor neuron diseases,alsin, an exchange factor for Rab5A [14], senataxin, potentiallyinvolved in RNA processing, VAPB protein regulating vesicle transport,the major axonal retrograde motor protein dynactin, mitochondrial genesfor cytochrome c oxidase and isoleucine tRNA synthetase [15,16],angiogenic modulators VEGF and angiogenin [17], can be mentioned.

ALS is a complex disease with multiple causes and the precise mechanismsinvolved in the pathogenesis of this disease are not yet resolved. Thischallenges the discovery of effective pharmacologic therapies. Clinicaltrials had shown that survival, but not function, is modestly prolongedby riluzole in ALS [18]. Nevertheless, riluzole is currently the onlydrug approved and the only known therapy for ALS. Regarding to theseverity of disease, it is consequently administered as adisease-modifying compound to all the patients suffering from ALS.

WO 2009/133128, WO 2009/133141, WO 2009/133142, WO 2011/054759, WO2009/068668 and WO 2009/153291 disclose potential treatments for severalneurodegenerative diseases, among which ALS.

First attempts at establishing guidelines for non-pharmacologicaltherapies were performed. However, standards are still based on expertopinion and differ between countries.

Accordingly, there is still a strong need in the art for novel andeffective therapies for treating ALS.

SUMMARY OF INVENTION

The present invention provides novel compositions and methods fortreating ALS and related disorders. The invention stems, inter alia,from the identification of drug combinations which provide improvedtherapeutic effect and clinical benefit to subjects having ALS.

More particularly, an object of the invention relates to a compositionfor use in the treatment of ALS or a related disorder, comprising atleast two drugs selected from acamprosate, baclofen, cinacalcet,mexiletine, sulfisoxazole and torasemide, or a salt, prodrug, derivativeof any chemical purity, or sustained-release formulation thereof.

A further object of the invention is a method for treating ALS or arelated disorder in a subject in need thereof, comprising administeringto the subject at least two drugs selected from acamprosate, baclofen,cinacalcet, mexiletine, sulfisoxazole and torasemide, or a salt,prodrug, derivative of any chemical purity, or sustained-releaseformulation thereof.

Preferred examples of drug combinations for use in the inventioninclude, e.g., baclofen and acamprosate; acamprosate and cinacalcet;torasemide and baclofen; baclofen and cinacalcet; torasemide andsulfisoxazole; mexiletine and cinacalcet; or baclofen and acamprosateand torasemide.

In a particular embodiment of the invention, the composition furthercomprises riluzole, or a salt, prodrug, derivative of any chemicalpurity, or sustained-release formulation thereof.

The compositions in the invention may further comprise one or severalpharmaceutically acceptable carrier(s) or excipient(s), and they may beadministered repeatedly to the subject. Preferred compositions areadministered orally. Moreover, the drugs may be formulated oradministered together, separately or sequentially.

The invention is suitable for treating ALS in any mammalian subject,particularly in a human subject, at any stage of the disease. Theinvention may be used to retard the development of the disease, toreduce, delay or prevent paralysis, motor neuron degeneration and/orpain, and/or to increase survival.

LEGEND TO THE FIGURES

FIG. 1: Effect of baclofen and acamprosate combination therapy againstglutamate toxicity on neuronal cortical cells. Glutamate intoxication issignificantly prevented by the combination of baclofen (400 nM) andacamprosate (1.6 nM) whereas, at those concentrations, baclofen andacamprosate alone have no significant effect on intoxication. *:p<0.001, significantly different from glutamate intoxication;(ANOVA+Dunnett Post-Hoc test).

FIGS. 2 A-C: Effect of baclofen and acamprosate combination therapyagainst glutamate (GLU) toxicity in the nerve-muscle cells co-culturemodel, on number (A), area (B), and neurite length (C) of motor units.Whatever the endpoint considered, glutamate intoxication issignificantly prevented by the combination of baclofen (BCL, 80 nM) andacamprosate (ACP, 0.32 nM), whereas, at those concentrations, baclofenand acamprosate alone have no significant effect on intoxication. *:p<0.05, significantly different from glutamate intoxication;(ANOVA+Dunnett Post-Hoc test).

FIG. 3: Effect of cinacalcet and mexiletine combination therapy againstglutamate toxicity on neuronal cortical cells. The glutamateintoxication is significantly prevented by the combination of cinacalcet(64 pM) and mexiletine (25.6 pM) whereas, at those concentrations,cinacalcet and mexiletine alone have no significant effect onintoxication. *: p<0.001, significantly different from glutamateintoxication; (ANOVA+Dunnett Post-Hoc test).

FIG. 4: Effect of sulfisoxazole and torasemide combination therapyagainst glutamate toxicity on neuronal cortical cells. The glutamateintoxication is significantly prevented by the combination ofsulfisoxazole (6.8 nM) and torasemide (400 nM) whereas, at thoseconcentrations, sulfisoxazole and torasemide alone have no significanteffect on intoxication. *: p<0.001, significantly different fromglutamate intoxication; (ANOVA+Dunnett Post-Hoc test).

FIGS. 5 A-B: Baclofen (BCL) and acamprosate (ACP) combination acts as anenhancer of riluzole (RIL) protecting effect against glutamate toxicity,in the nerve-muscle cells co-culture model, area (A), and neurite length(B) of motor units. A significantly stronger enhancing effect isobserved when using concentrations as low as ACP (0.14 nM) BCL (36 nM),when compared to the effect obtained for ACP (0.32 nM) BCL (80 nM). (*:p<0.001, significantly different from glutamate intoxication; ⋄:p<0.001, significantly different (ANOVA+Dunnett Post-Hoc test)).

FIGS. 6 A-B: Baclofen (BCL) and acamprosate (ACP) act synergisticallywith riluzole in protecting neuromuscular junctions against glutamatetoxicity in the nerve-muscle cells co-culture model. An importantimprovement of protection is observed when measuring the number, area,as well as neurite length of motor units. A) an improvement of endpointsfrom 2 to 5% is observed for ACP (0.14 nM) BCL (36 nM) mix; from 12 to16% when using riluzole (0.04 μM) alone; whereas the combination of the3 drugs results in an improvement from 39 to 43%. B) an improvement ofendpoints from 2 to 5% is observed for ACP (0.14 nM) BCL (36 nM) mix;from 70 to 88% when using riluzole 5 μM; whereas the combination of the3 drugs results in an improvement from 131 to 165%. (*: p<0.001,significantly different from glutamate intoxication; ⋄: p<0.001,significantly different (ANOVA+Dunnett Post-Hoc test)).

FIGS. 7 A-B: Co-incubation of baclofen-acamprosate combination withriluzole (without riluzole pre-treatment) improves the effect ofriluzole alone in protecting neuromuscular junctions against glutamatetoxicity in the nerve-muscle cells co-culture model. An importantimprovement of protection is observed when measuring the number andneurite length of motor units. A) The compositions of the inventionsignificantly improve the beneficial effect of riluzole at 5 μM on motorunit numbers. B) The compositions of the invention significantly improvethe effect of riluzole at 0.04 and 5 μM on neurite length. (*: p<0.05,significantly different from glutamate toxicity; (Dunnett Post-Hoctest)). White bar: control; black bar: glutamate treatment (60 μM);light grey bar: riluzole treatment (0.04 nM); dark grey bar: riluzoletreatment (5 μM); diagonally dashed bars: treatment with combination ofacamprosate and baclofen at 0.14 nM and 32 nM, respectively;horizontally dashed bars: treatment with combination of acamprosate andbaclofen at 0.32 nM and 80 nM, respectively.

FIGS. 8 A-C: Switching from riluzole treatment to baclofen-acamprosatetreatment results in an improved efficiency in the neuromuscularjunctions protection as compared to i) riluzole and ii)baclofen-acamprosate therapies. Riluzole (5 μM) is applied for 96 hours,removed and then treatment with the compositions of the invention isapplied. A) Riluzole pre-treatment followed by the treatment withbaclofen and acamprosate combination improves motor unit area in theglutamate toxicity model. B) Riluzole pre-treatment followed by thetreatment with baclofen and acamprosate combination improves motor unitnumber in the glutamate toxicity model. C) Riluzole pre-treatmentfollowed by the treatment with baclofen and acamprosate combinationimproves neurite length in the glutamate toxicity model. (*: p<0.05,significantly different from glutamate toxicity; (Dunnett Post-Hoctest)). White bar: control; black bar: glutamate treatment (60 μM); greybar: riluzole treatment (5 μM); diagonally dashed bars: treatment withcombination of acamprosate and baclofen at 0.14 nM and 32 nM,respectively; horizontally dashed bars: treatment with combination ofacamprosate and baclofen at 0.32 nM and 80 nM, respectively.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides new therapeutic approaches for treatingALS or related disorders. More particularly, the present inventiondiscloses novel combinatorial therapies which allow an effectivecorrection of such diseases and may be used in any mammalian subject.

Within the context of this invention, the term “treatment” of a disorderincludes the therapy, prophylaxis, retardation or reduction of painprovoked by the disorder. The term treatment includes in particular thecontrol of disease progression and associated symptoms. In relation toALS, the term treatment also designates a retardation or delayed onsetof paralysis, a reduction or prevention of motor neuron degeneration, areduction of pain, and/or an increase in survival.

The term “ALS related disorders” refers to motor neuron disorders asprimary lateral sclerosis (PLS), progressive muscular atrophy (PMA),pseudobulbar palsy and progressive bulbar palsy (PBP), as well as tofronto temporal dementia (FTD).

The term “combination” or “combinatorial treating/therapy” designates atreatment wherein at least two or more drugs are co-administered to asubject to cause a biological effect. In a combined therapy according tothis invention, the at least two drugs may be administered together orseparately, at the same time or sequentially. Also, the at least twodrugs may be administered through different routes and protocols. As aresult, although they may be formulated together, the drugs of acombination may also be formulated separately.

As discussed above, the invention relates to drugs compositions andmethods for treating ALS or a related disorder in a subject in needthereof.

More particularly, the invention relates to a composition for use in thetreatment of ALS or a related disorder, comprising at least two drugsselected from the group consisting of acamprosate, baclofen, cinacalcet,mexiletine, sulfisoxazole and torasemide, or salts or prodrugs orderivatives of any purity or sustained release formulations thereof.

The inventors have surprisingly found that these compounds, incombination(s), show a protective activity against glutamate toxicity,which is one of the causes of neuronal death, in an in vitro model forALS.

More particularly, the invention shows that drugs of the invention exerta surprising protective activity on motor neurons against glutamatetoxicity, which is one of the etiological causes of nerve degenerationin ALS. Moreover, the inventors have observed that these compounds, atlow doses, act synergistically to efficiently protect motor units. Thisis a particularly substantial advantage, which avoids any potential sideeffects. Moreover, as shown in the experimental part, these combinationtherapies can delay the onset of paralysis in vivo in animal models ofALS, and prolong life duration. These combination therapies thereforerepresent substantial improvement in the treatment of ALS subjects.

Accordingly, the invention relates to a composition for use in thetreatment of ALS or a related disorder, comprising at least two drugsselected from acamprosate, baclofen, cinacalcet, mexiletine,sulfisoxazole and torasemide, or salt(s) or prodrug(s) or derivative(s)of any purity or sustained release formulations thereof.

The invention also relates to a method for treating ALS or a relateddisorder in a subject in need thereof, comprising administering to thesubject at least two drugs selected from the group consisting ofacamprosate, baclofen, cinacalcet, mexiletine, sulfisoxazole andtorasemide, or salt(s) or prodrug(s) or derivative(s) of any purity orsustained release formulations thereof.

The invention also relates to a compound selected from acamprosate,baclofen, cinacalcet, mexiletine, sulfisoxazole and torasemide, orsalt(s) or prodrug(s) or derivative(s) of any purity or sustainedrelease formulations thereof, in combination with at least one secondcompound selected from acamprosate, baclofen, cinacalcet, mexiletine,sulfisoxazole and torasemide, or salt(s) or prodrug(s) or derivative(s)of any purity or sustained release formulations thereof, for use in thetreatment of ALS or a related disorder by combined, separate orsequential administration to a subject in need thereof.

Preferred combinations for use in the present invention comprise atleast one of the following drug combinations, for simultaneous,sequential or separate administration:

-   -   baclofen and cinacalcet,    -   cinacalcet and acamprosate,    -   baclofen and acamprosate,    -   baclofen and acamprosate and torasemide,    -   mexiletine and cinacalcet,    -   torasemide and baclofen, or    -   torasemide and sulfisoxazole,

or salt(s) or prodrug(s) or derivative(s) of any purity or sustainedrelease formulations thereof for use in the treatment of ALS or arelated disorder. Compositions comprising such combinations alsorepresent specific objects of the invention.

The term “prodrug” as used herein refers to any functional derivatives(or precursors) of a compound of the present invention, which, whenadministered to a biological system (e.g. a human organism), generatessaid compound as a result of e.g., spontaneous chemical reaction(s),enzyme catalysed chemical reaction(s), and/or metabolic chemicalreaction(s). Prodrugs typically have the structure X-drug, wherein X isan inert carrier moiety and drug is the active compound. Prodrugs areusually inactive or less active than the resulting drug and can be used,for example, to improve the physicochemical properties of the drug, totarget the drug to a specific tissue, to improve the pharmacokinetic andpharmacodynamic properties of the drug and/or to reduce undesirable sideeffects. Some of the common functional groups that are amenable toprodrug design include, but are not limited to, carboxylic, hydroxyl,amine, phosphate/phosphonate and carbonyl groups. Prodrugs typicallyproduced via the modification of these groups include, but are notlimited to, esters, carbonates, carbamates, amides and phosphates.Specific technical guidance for the selection of suitable prodrugs isgeneral common knowledge [19-23]. Furthermore, the preparation ofprodrugs may be performed by conventional methods known by those skilledin the art. Methods which can be used to synthesize other prodrugs aredescribed in numerous reviews on the subject [20,24-30]. For example,arbaclofen placarbil is listed in ChemID plus Advance database (website:chem.sis.nlm.nih.gov/chemidplus/) and arbaclofen placarbil is awell-known prodrug of baclofen [31,32]. Specific examples of prodrugs ofbaclofen are given in Hanafi et al., 2011 [33], particularly baclofenesters and baclofen ester carbamates, which are of particular interestfor CNS targeting. Hence such prodrugs are particularly suitable forcompositions of this invention. Arbaclofen placarbil as mentioned beforeis also a well-known prodrug and may thus be used instead of baclofen incompositions of the invention. Other prodrugs of baclofen can be foundin the following patent applications: WO 2010/102071, US 2009/197958, WO2009/096985, WO 2009/061934, WO 2008/086492, US 2009/216037, WO2005/066122, US 2011/021571, WO 2003/077902 and WO 2010/120370.

Useful prodrugs for acamprosate such as pantoic acid ester neopentylsulfonyl esters, neopentyl sulfonyl esters prodrugs or maskedcarboxylate neopentyl sulfonyl ester prodrugs of acamprosate are notablylisted in WO 2009/033069, WO 2009/033061, WO 2009/033054 WO 2009/052191,WO 2009/033079, US 2009/0099253, US 2009/0069419, US 2009/0082464, US2009/0082440 and US 2009/0076147.

The term “derivative” of a compound includes any molecule that isfunctionally and/or structurally related to said compound, such as anacid, amide, ester, ether, acetylated variant, hydroxylated variant, oran alkylated (C1-C6) variant of such a compound. The term derivativealso includes structurally related compound having lost one or moresubstituent as listed above. For example, homotaurine is a deacetylatedderivative of acamprosate. Preferred derivatives of a compound aremolecules having a substantial degree of similarity to said compound, asdetermined by known methods. Similar compounds along with their index ofsimilarity to a parent molecule can be found in numerous databases suchas PubChem (http://pubchem.ncbi.nlm.nih.gov/search/) or DrugBank(http://www.drugbank.ca/). In a more preferred embodiment, derivativesshould have a Tanimoto similarity index greater than 0.4, preferablygreater than 0.5, more preferably greater than 0.6, even more preferablygreater than 0.7 with a parent drug. The Tanimoto similarity index iswidely used to measure the degree of structural similarity between twomolecules. Tanimoto similarity index can be computed by software such asthe Small Molecule Subgraph Detector [34,35] available online(http://www.ebi.ac.uk/thornton-srv/software/SMSD/). Preferredderivatives should be both structurally and functionally related to aparent compound, i.e., they should also retain at least part of theactivity of the parent drug, more preferably they should show aprotective activity against glutamate toxicity for the motor units (asexemplified in the experimental part).

The term derivatives also include metabolites of a drug, e.g., amolecule which results from the (biochemical) modification(s) orprocessing of said drug after administration to an organism, usuallythrough specialized enzymatic systems, and which displays or retains abiological activity of the drug. Metabolites have been disclosed asbeing responsible for much of the therapeutic action of the parent drug.In a specific embodiment, a “metabolite” as used herein designates amodified or processed drug that retains at least part of the activity ofthe parent drug, more preferably they should show a protective activityagainst glutamate toxicity for the motor units (as exemplified in theexperimental part). Examples of metabolites include hydroxylated formsof torasemide resulting from the hepatic metabolism of the drug (Drugbank database [36]).

The term “salt” refers to a pharmaceutically acceptable and relativelynon-toxic, inorganic or organic acid addition salt of a compound of thepresent invention. Pharmaceutical salt formation consists in pairing anacidic, basic or zwitterionic drug molecule with a counterion to createa salt version of the drug. A wide variety of chemical species can beused in neutralization reaction. Pharmaceutically acceptable salts ofthe invention thus include those obtained by reacting the main compound,functioning as a base, with an inorganic or organic acid to form a salt,for example, salts of acetic acid, nitric acid, tartric acid,hydrochloric acid, sulfuric acid, phosphoric acid, methane sulfonicacid, camphor sulfonic acid, oxalic acid, maleic acid, succinic acid orcitric acid. Pharmaceutically acceptable salts of the invention alsoinclude those in which the main compound functions as an acid and isreacted with an appropriate base to form, e.g., sodium, potassium,calcium, magnesium, ammonium, or choline salts. Though most of salts ofa given active principle are bioequivalents, some may have, amongothers, increased solubility or bioavailability properties. Saltselection is now a common standard operation in the process of drugdevelopment as taught by H. Stahl and C. G Wermuth in their handbook[37].

In a preferred embodiment, the designation of a compound is meant todesignate the compound per se, as well as any pharmaceuticallyacceptable salt, hydrate, isomer, or racemate thereof.

Table 1 below provides CAS number of compounds for use in the invention,as well as of salt(s), derivative(s), metabolite(s), and/or prodrug(s)of the compounds.

TABLE 1 Class or Tanimoto Drug CAS Numbers similarity index acamprosateand related compounds acamprosate 77337-76-9; 77337-73-6 NA homotaurine3687-18-1 0.73 ethyl dimethyl ammonio / 0.77 propane sulfonate taurine107-35-7 0.5  baclofen and related compounds baclofen 1134-47-0;66514-99-6; NA 69308-37-8; 70206-22-3; 63701-56-4; 63701-55-33-(p-chlorophenyl)-4- / Metabolite hydroxybutyric acid arbaclofenplacarbil 847353-30-4 Prodrug mexiletine and related compoundsmexiletine 31828-71-4; 5370-01-4 6-hydroxymethylmexiletine 53566-98-6Metabolite 4-hydroxymexiletine 53566-99-7 Metabolite 3-hydroxymexiletine(MHM) 129417-37-4 Metabolite N-hydroxymexiletine 151636-18-9 Metaboliteglucuronide sulfisoxazole and related compounds sulfisoxazole 127-69-5;4299-60-9 N(4)-acetylsulfisoxazole 4206-74-0 Metabolite sulfisoxazoleacetyl 80-74-0 Prodrug sulfamethoxazole 723-46-6 0.52 cinacalcet andrelated compounds cinacalcet 226256-56-0; 364782-34-3 hydrocinnamic acid501-52-0 Metabolite torasemide and related compounds torasemide56211-40-6; 72810-59-4 hydroxytorasemide 99300-68-2; 99300-67-1Metabolites carboxytorasemide Metabolite tolbutamide 64-77-7 0.55

More preferably, drug compositions of the invention comprise 2, 3, 4 or5 distinct drugs, even more preferably 2, 3 or 4 distinct drugs forcombinatorial treatment of ALS or a related disorder in a subject inneed thereof.

In a particular embodiment, the invention relates composition per secomprising baclofen and cinacalcet, or acamprosate and cinacalcet orsalt(s) or prodrug(s) or derivative(s) of any purity or sustainedrelease formulations.

In a more particular embodiment, the invention relates to a compositioncomprising acamprosate and cinaclacet or salt(s) or prodrug(s) orderivative(s) of any purity or sustained release formulations for itsuse in the treatment of ALS or a related disorder, wherein the dailydosage of acamprosate is equal or lower to 10 mg.

Furthermore, in another particular embodiment, the compositions andmethods of the invention further use riluzole, or a salt, prodrug,derivative of any purity, or sustained release formulation thereof. Theresults presented indeed surprisingly show that, when used incombination with riluzole (CAS No. 1744-22-5), compositions of theinvention can substantially increase the clinical benefit of thetreatment to patients.

Accordingly, a particular object of this invention is a compositioncomprising i) at least one drug selected from acamprosate, baclofen,cinacalcet, mexiletine, sulfisoxazole and torasemide, and ii) riluzole,for a simultaneous, separate or sequential use in the treatment of ALSor a related disorder.

Another particular object of this invention is a composition asdisclosed above, comprising i) at least two drugs selected fromacamprosate, baclofen, cinacalcet, mexiletine, sulfisoxazole andtorasemide, and ii) riluzole, for a simultaneous, separate or sequentialuse in the treatment of ALS or a related disorder.

A preferred object of this invention is a composition comprising atleast one of the following drug combinations:

-   -   riluzole, baclofen and cinacalcet,    -   riluzole, cinacalcet and acamprosate,    -   riluzole, baclofen and acamprosate,    -   riluzole, baclofen and acamprosate and torasemide,    -   riluzole, mexiletine and cinacalcet,    -   riluzole, torasemide and baclofen, or    -   riluzole, torasemide and sulfisoxazole,

or salt(s) or prodrug(s) or derivative(s) of any purity or sustainedrelease formulations thereof, for a simultaneous, separate or sequentialuse in the treatment of ALS or a related disorder.

Other additional therapies that can be used in conjunction with drugcombination(s) according to the present invention, may comprise one ormore drug(s) that ameliorate(s) symptoms of ALS, one or more drug(s)that could be used for palliative treatment of ALS or one or moredrug(s) currently evaluated in the frame of clinical trials for treatingof ALS. Preferably, said one or more drug(s) is/are selected from AEOL10150, arimoclomol, AVP-923, botulinum toxin type B (Myobloc),ceftriaxone, celastrol, celecoxib, cistanche total glycosides, coenzymeQ10, copaxone, creatine, creatinine, dronabinol, erythropoietin,escitalopram (Lexapro), glatiramer acetate, granulocyte-colonystimulating factor (G-CSF), growth hormone (Somatropin), GSK1223249,indinavir, insulin-like growth factor-1 (IGF-I), IGF-1-AAV, KNS-760704,leteprinim, leuprolide, levetiracetam, MCI-186, mecobalamin,minocycline, modafinil, Naaladase inhibitor, N-Acetylcysteine, NBQX,nimesulide, nimodipine, olanzapine, olesoxime (TRO19622), ONO-2506,oxepa, pioglitazone, R(+) pramipexole dihydrochloride monohydrate,olesoxime, oxandrolone, quinidine, phenyl butyrate, SB-509, Scriptaid,sNN0029, somatropine, talampanel, tamoxifen, tauroursodeoxycholic acid,TCH346, testosterone, thalidomide, trehalose, tretinoin, vitamin E,YAM80 or from 17-beta-estradiol, 2-MPPA(2-(3-mercaptopropyl)pentanedioic acid), 3,4-diaminopyridine,5-hydroxytryptophan, 7-nitroindazole, alpha-lipoic acid, AM1241,aminophylline, angiogenin, anti-human SOD1 antibody, antisense peptidenucleic acid directed against p75(NTR), AP7, apocynin, BAPTA-AM, BDNF,BN82451, cannabinol, cardiotrophin-1, CD4 antibodies, CNTF, colivelin,dietary copper, corticotrophin, cyclophosphamide,Delta(9)-tetrahydrocannabinol, DHEA, diazepam, dietary zinc, diltiazem,DMPO, DP-109, DP-460, edaravone, EGCG, epigallocatechin gallate,etidronate, FeTCPP, fluvoxamine, folic acid, gabapentin, galectin-1,GDNF, ginseng, GPI-1046, guanidine, HGF, humanin, IFN-alpha,interleukin-3, ivermectin, L-745,870, L-carnitine, L-DOPA, lecithinizedSOD, lenalidomide, leupeptin, LIF, L-NAME, lysine acetylsalicylate,melatonin, mepivacaine, methamphetamine, methylcobalamin, MK-801,MnTBAP, modafinil, morphine, Neu2000, NGF, nordihydroguaiaretic acid,nortriptyline, NT3, olmesartan, penicillamine, pentoxifylline, pimozide,polyamine-modified catalase, pramipexole, prednisone, progesterone,promethazine, putrescine-modified catalase, pyruvate, rasagiline, RK35,Ro 28-2653, rofecoxib, RPR 119990, RX77368, SB203580, selegiline,semapimod, sertraline, SS-31, SSR180575, stabilized siRNA against humanCu,Zn-superoxide dismutase (SOD1), tacrolimus, tamsulosin hydrochloride,TAT-modified Bcl-X(L), TGF-beta2, tianeptine, trientine, TR019622,U-74389F, VEGF, vincristine, WHI-P131, WIN55, 212-2, WX-340, xaliproden,ZK 187638 and zVAD-fmk.

As indicated above, the preferred therapies of this invention contains2, 3, 4 or even more distinct active compounds, which may optionally befurther associated or combined with other treatment(s). In a combinationtherapy of this invention, the compounds or drugs may be formulatedtogether or separately, and administered together, separately orsequentially.

The invention also relates to a method of treating ALS disease or arelated disorder, the method comprising simultaneously, separately orsequentially administering to a subject in need thereof a drugcombination as disclosed above.

The drugs or compositions of the invention may be administeredrepeatedly to the subject.

The compositions of the invention typically comprise one or severalpharmaceutically acceptable carriers or excipients.

A further object of this invention relates to the use of at least twodrugs selected from the group consisting of acamprosate, baclofen,cinacalcet, mexiletine, sulfisoxazole and torasemide for the manufactureof a medicament for the treatment of ALS or a related disorder bycombined, separate or sequential administration to a subject in needthereof.

A further object of this invention relates to the use of at least one ofthe following drug combinations:

-   -   baclofen and cinacalcet,    -   cinacalcet and acamprosate,    -   baclofen and acamprosate,    -   baclofen and acamprosate and torasemide,    -   mexiletine and cinacalcet,    -   torasemide and baclofen, or    -   torasemide and sulfisoxazole,

for the manufacture of a medicament for the treatment of ALS or arelated disorder by combined, separate or sequential administration to asubject in need thereof.

In a particular embodiment, the invention also relates to the use of atleast one drug selected from the group consisting of acamprosate,baclofen, cinacalcet, mexiletine, sulfisoxazole and torasemide in acombination with the riluzole for the manufacture of a medicament forthe treatment of ALS or a related disorder by combined, separate orsequential administration to a subject in need thereof.

Another embodiment relates to the use of at least two drugs selectedfrom the group consisting of acamprosate, baclofen, cinacalcet,mexiletine, sulfisoxazole and torasemide in a combination with theriluzole for the manufacture of a medicament for the treatment of ALS ora related disorder by combined, separate or sequential administration toa subject in need thereof.

In a more particular embodiment, of this invention relates to the use ofat least one of the following drug combinations:

-   -   baclofen and cinacalcet,    -   cinacalcet and acamprosate,    -   baclofen and acamprosate,    -   baclofen and acamprosate and torasemide,    -   mexiletine and cinacalcet,    -   torasemide and baclofen, or    -   torasemide and sulfisoxazole,

in a combination with riluzole, for the manufacture of a medicament forthe treatment of ALS or a related disorder by combined, separate orsequential administration to a subject in need thereof.

A further object of this invention is a method of preparing apharmaceutical composition, the method comprising mixing the abovecompounds in an appropriate excipient or carrier.

Therapy according to the invention may be provided at home, the doctor'soffice, a clinic, a hospital's outpatient department, or a hospital, sothat the doctor can observe the therapy's effects closely and make anyadjustments that are needed.

The duration of the therapy depends on the stage of the disease, the ageand condition of the patient, and how the patient responds to thetreatment.

Additionally, a person having a greater risk of developing an additionalneuropathic disorder (e.g., a person who is genetically predisposed toor have, for example, diabetes, or is being under treatment for anoncological condition, etc.) may receive prophylactic treatment toalleviate or to delay eventual neuropathic response.

The dosage, frequency and mode of administration of each drug can becontrolled independently. Combination therapy may be given in on-and-offcycles that include rest periods so that the patient's body has a chanceto recovery from any as yet unforeseen side-effects. The drugs may alsobe formulated together such that one administration delivers both drugs.

Formulation of Pharmaceutical Compositions

The administration of each drug of the combination may be by anysuitable means that results in a concentration of the drug that,combined with the other component, is able to ameliorate the patientcondition.

While it is possible for the active ingredients of the combination to beadministered as the pure chemical it is preferable to present them as apharmaceutical composition, also referred to in this context aspharmaceutical formulation. Possible compositions include those suitablefor oral, rectal, topical (including transdermal, buccal andsublingual), or parenteral (including subcutaneous, intramuscular,intravenous and intradermal) administration.

More commonly these pharmaceutical formulations are prescribed to thepatient in “patient packs” containing a number dosing units or othermeans for administration of metered unit doses for use during a distincttreatment period in a single package, usually a blister pack. Patientpacks have an advantage over traditional prescriptions, where apharmacist divides a patient's supply of a pharmaceutical from a bulksupply, in that the patient always has access to the package insertcontained in the patient pack, normally missing in traditionalprescriptions. The inclusion of a package insert has been shown toimprove patient compliance with the physician's instructions. Thus, theinvention further includes a pharmaceutical formulation, as hereinbefore described, in combination with packaging material suitable forsaid formulations. In such a patient pack the intended use of aformulation for the combination treatment can be inferred byinstructions, facilities, provisions, adaptations and/or other means tohelp using the formulation most suitably for the treatment. Suchmeasures make a patient pack specifically suitable for and adapted foruse for treatment with the combination of the present invention.

The drug may be contained in any appropriate amount in any suitablecarrier substance, and is may be present in an amount of 1-99% by weightof the total weight of the composition. The composition may be providedin a dosage form that is suitable for the oral, parenteral (e.g.,intravenously, intramuscularly), rectal, cutaneous, nasal, vaginal,inhalant, skin (patch), or ocular administration route. Thus, thecomposition may be in the form of, e.g., tablets, capsules, pills,powders, granulates, suspensions, emulsions, solutions, gels includinghydrogels, pastes, ointments, creams, plasters, drenches, osmoticdelivery devices, suppositories, enemas, injectables, implants, sprays,or aerosols.

The pharmaceutical compositions may be formulated according toconventional pharmaceutical practice (see, e.g., Gennaro [38] and theEncyclopedia of Pharmaceutical Technology [39]).

Pharmaceutical compositions according to the invention may be formulatedto release the active drug substantially immediately upon administrationor at any predetermined time or time period after administration.

The controlled release formulations include (i) formulations that createa substantially constant concentration of the drug within the body overan extended period of time; (ii) formulations that after a predeterminedlag time create a substantially constant concentration of the drugwithin the body over an extended period of time; (iii) formulations thatsustain drug action during a predetermined time period by maintaining arelatively, constant, effective drug level in the body with concomitantminimization of undesirable side effects associated with fluctuations inthe plasma level of the active drug substance; (iv) formulations thatlocalize drug action by, e.g., spatial placement of a controlled releasecomposition adjacent to or in the diseased tissue or organ; and (v)formulations that target drug action by using carriers or chemicalderivatives to deliver the drug to a particular target cell type.

Administration of drugs in the form of a controlled release formulationis especially preferred in cases in which the drug in combination, has(i) a narrow therapeutic index (i.e., the difference between the plasmaconcentration leading to harmful side effects or toxic reactions and theplasma concentration leading to a therapeutic effect is small; ingeneral, the therapeutic index, TI, is defined as the ratio of medianlethal dose (LD50) to median effective dose (ED50)); (ii) a narrowabsorption window in the gastro-intestinal tract; or (iii) a very shortbiological half-life so that frequent dosing during a day is required inorder to sustain the plasma level at a therapeutic level.

Any of a number of strategies can be pursued in order to obtaincontrolled release in which the rate of release outweighs the rate ofmetabolism of the drug in question. Controlled release may be obtainedby appropriate selection of various formulation parameters andingredients, including, e.g., various types of controlled releasecompositions and coatings. Thus, the drug is formulated with appropriateexcipients into a pharmaceutical composition that, upon administration,releases the drug in a controlled manner (single or multiple unit tabletor capsule compositions, oil solutions, suspensions, emulsions,microcapsules, microspheres, nanoparticles, patches, and liposomes).

Solid Dosage Forms for Oral Use

Preferred administration route for cilostazol and for riluzole is theoral route. Formulations for oral use include tablets containing theactive ingredient(s) in a mixture with non-toxic pharmaceuticallyacceptable excipients. These excipients may be, for example, inertexcipients or fillers (e.g., sucrose, microcrystalline cellulose,starches including potato starch, calcium carbonate, sodium chloride,calcium phosphate, calcium sulfate, or sodium phosphate); granulatingand disintegrating agents (e.g., cellulose derivatives includingmicrocrystalline cellulose, starches including potato starch,croscarmellose sodium, alginates, or alginic acid); binding agents(e.g., acacia, alginic acid, sodium alginate, gelatin, starch,pregelatinized starch, microcrystalline cellulose,carboxymethylcellulose sodium, methylcellulose, hydroxypropylmethylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethyleneglycol); and lubricating agents, glidants, and antiadhesives (e.g.,stearic acid, silicas, or talc). Other pharmaceutically acceptableexcipients can be colorants, flavoring agents, plasticizers, humectants,buffering agents, and the like.

The tablets may be uncoated or they may be coated by known techniques,optionally to delay disintegration and absorption in thegastrointestinal tract and thereby providing a sustained action over alonger period. The coating may be adapted to release the active drugsubstance in a predetermined pattern (e.g., in order to achieve acontrolled release formulation) or it may be adapted not to release theactive drug substance until after passage of the stomach (entericcoating). The coating may be a sugar coating, a film coating (e.g.,based on hydroxypropyl methylcellulose, methyl cellulose, methylhydroxyethyl cellulose, hydroxypropyl cellulose, carboxymethylcellulose,acrylate copolymers, polyethylene glycols and/or polyvinylpyrrolidone),or an enteric coating (e.g., based on methacrylic acid copolymer,cellulose acetate phthalate, hydroxypropyl methylcellulose phthalate,hydroxypropyl methylcellulose acetate succinate, polyvinyl acetatephthalate, shellac, and/or ethylcellulose). A time delay material suchas, e.g., glyceryl monostearate or glyceryl distearate may be employed.

The solid tablet compositions may include a coating adapted to protectthe composition from unwanted chemical changes, (e.g., chemicaldegradation prior to the release of the active drug substance). Thecoating may be applied on the solid dosage form in a similar manner asthat described in Encyclopedia of Pharmaceutical Technology [39].

The drugs may be mixed together in the tablet, or may be partitioned.For example, a first drug is contained on the inside of the tablet, anda second drug is on the outside, such that a substantial portion of thesecond drug is released prior to the release of the first drug.

Formulations for oral use may also be presented as chewable tablets, oras hard gelatin capsules wherein the active ingredient is mixed with aninert solid excipient (e.g., potato starch, microcrystalline cellulose,calcium carbonate, calcium phosphate or kaolin), or as soft gelatincapsules wherein the active ingredient is mixed with water or an oilmedium, for example, liquid paraffin, or olive oil. Powders andgranulates may be prepared using the ingredients mentioned above undertablets and capsules in a conventional manner.

Controlled release compositions for oral use may, e.g., be constructedto release the active drug by controlling the dissolution and/or thediffusion of the active drug substance.

Dissolution or diffusion controlled release can be achieved byappropriate coating of a tablet, capsule, pellet, or granulateformulation of drugs, or by incorporating the drug into an appropriatematrix. A controlled release coating may include one or more of thecoating substances mentioned above and/or, e.g., shellac, beeswax,glycowax, castor wax, carnauba wax, stearyl alcohol, glycerylmonostearate, glyceryl distearate, glycerol palmitostearate,ethylcellulose, acrylic resins, dl-polylactic acid, cellulose acetatebutyrate, polyvinyl chloride, polyvinyl acetate, vinyl pyrrolidone,polyethylene, polymethacrylate, methylmethacrylate,2-hydroxymethacrylate, methacrylate hydrogels, 1,3 butylene glycol,ethylene glycol methacrylate, and/or polyethylene glycols. In acontrolled release matrix formulation, the matrix material may alsoinclude, e.g., hydrated methylcellulose, carnauba wax and stearylalcohol, carbopol 934, silicone, glyceryl tristearate, methylacrylate-methyl methacrylate, polyvinyl chloride, polyethylene, and/orhalogenated fluorocarbon.

A controlled release composition containing one or more of the drugs ofthe claimed combinations may also be in the form of a buoyant tablet orcapsule (i.e., a tablet or capsule that, upon oral administration,floats on top of the gastric content for a certain period of time). Abuoyant tablet formulation of the drug(s) can be prepared by granulatinga mixture of the drug(s) with excipients and 20-75% w/w ofhydrocolloids, such as hydroxyethylcellulose, hydroxypropylcellulose, orhydroxypropyl-methylcellulose. The obtained granules can then becompressed into tablets. On contact with the gastric juice, the tabletforms a substantially water-impermeable gel barrier around its surface.This gel barrier takes part in maintaining a density of less than one,thereby allowing the tablet to remain buoyant in the gastric juice.

Liquids for Oral Administration

Powders, dispersible powders, or granules suitable for preparation of anaqueous suspension by addition of water are convenient dosage forms fororal administration. Formulation as a suspension provides the activeingredient in a mixture with a dispersing or wetting agent, suspendingagent, and one or more preservatives. Suitable suspending agents are,for example, sodium carboxymethylcellulose, methylcellulose, sodiumalginate, and the like.

Parenteral Compositions

Although less preferred, the pharmaceutical composition may also beadministered parenterally by injection, infusion or implantation(intravenous, intramuscular, subcutaneous, or the like) in dosage forms,formulations, or via suitable delivery devices or implants containingconventional, non-toxic pharmaceutically acceptable carriers andadjuvants. The formulation and preparation of such compositions are wellknown to those skilled in the art of pharmaceutical formulation.

Compositions for parenteral use may be provided in unit dosage forms(e.g., in single-dose ampoules), or in vials containing several dosesand in which a suitable preservative may be added (see below). Thecomposition may be in form of a solution, a suspension, an emulsion, aninfusion device, or a delivery device for implantation or it may bepresented as a dry powder to be reconstituted with water or anothersuitable vehicle before use. Apart from the active drug(s), thecomposition may include suitable parenterally acceptable carriers and/orexcipients. The active drug(s) may be incorporated into microspheres,microcapsules, nanoparticles, liposomes, or the like for controlledrelease. The composition may include suspending, solubilizing,stabilizing, pH-adjusting agents, and/or dispersing agents.

The pharmaceutical compositions according to the invention may be in theform suitable for sterile injection. To prepare such a composition, thesuitable active drug(s) are dissolved or suspended in a parenterallyacceptable liquid vehicle. Among acceptable vehicles and solvents thatmay be employed are water, water adjusted to a suitable pH by additionof an appropriate amount of hydrochloric acid, sodium hydroxide or asuitable buffer, 1,3-butanediol, Ringer's solution, and isotonic sodiumchloride solution. The aqueous formulation may also contain one or morepreservatives (e.g., methyl, ethyl or n-propyl p-hydroxybenzoate). Incases where one of the drugs is only sparingly or slightly soluble inwater, a dissolution enhancing or solubilizing agent can be added, orthe solvent may include 10-60% w/w of propylene glycol or the like.

Controlled release parenteral compositions may be in form of aqueoussuspensions, microspheres, microcapsules, magnetic microspheres, oilsolutions, oil suspensions, or emulsions. Alternatively, the activedrug(s) may be incorporated in biocompatible carriers, liposomes,nanoparticles, implants, or infusion devices. Materials for use in thepreparation of microspheres and/or microcapsules are, e.g.,biodegradable/bioerodible polymers such as polygalactin, poly-(isobutylcyanoacrylate), poly(2-hydroxyethyl-L-glutamnine). Biocompatiblecarriers that may be used when formulating a controlled releaseparenteral formulation are carbohydrates (e.g., dextrans), proteins(e.g., albumin), lipoproteins, or antibodies. Materials for use inimplants can be non-biodegradable (e.g., polydimethyl siloxane) orbiodegradable (e.g., poly(caprolactone), poly(glycolic acid) orpoly(ortho esters)).

Alternative Routes

Although less preferred and less convenient, other administrationroutes, and therefore other formulation, may be contemplated. In thisregard, for rectal application, suitable dosage forms for a compositioninclude suppositories (emulsion or suspension type), and rectal gelatincapsules (solutions or suspensions). In a typical suppositoryformulation, the active drug(s) are combined with an appropriatepharmaceutically acceptable suppository base such as cocoa butter,esterified fatty acids, glycerinated gelatin, and various water-solubleor dispersible bases like polyethylene glycols. Various additives,enhancers, or surfactants may be incorporated.

The pharmaceutical compositions may also be administered topically onthe skin for percutaneous absorption in dosage forms or formulationscontaining conventionally non-toxic pharmaceutical acceptable carriersand excipients including microspheres and liposomes. The formulationsinclude creams, ointments, lotions, liniments, gels, hydrogels,solutions, suspensions, sticks, sprays, pastes, plasters, and otherkinds of transdermal drug delivery systems. The pharmaceuticallyacceptable carriers or excipients may include emulsifying agents,antioxidants, buffering agents, preservatives, humectants, penetrationenhancers, chelating agents, gel-forming agents, ointment bases,perfumes, and skin protective agents.

The emulsifying agents may be naturally occurring gums (e.g., gum acaciaor gum tragacanth).

The preservatives, humectants, penetration enhancers may be parabens,such as methyl or propyl p-hydroxybenzoate, and benzalkonium chloride,glycerin, propylene glycol, urea, etc.

The pharmaceutical compositions described above for topicaladministration on the skin may also be used in connection with topicaladministration onto or close to the part of the body that is to betreated. The compositions may be adapted for direct application or forapplication by means of special drug delivery devices such as dressingsor alternatively plasters, pads, sponges, strips, or other forms ofsuitable flexible material.

Dosages and Duration of the Treatment

It will be appreciated that the drugs of the combination may beadministered concomitantly, either in the same or differentpharmaceutical formulation or sequentially. If there is sequentialadministration, the delay in administering one of the active ingredientsshould not be such as to lose the benefit of the efficacious effect ofthe combination of the active ingredients. A minimum requirement for acombination according to this description is that the combination shouldbe intended for combined use with the benefit of the efficacious effectof the combination of the active ingredients. The intended use of acombination can be inferred by facilities, provisions, adaptationsand/or other means to help using the combination according to theinvention.

One remarkable advantage of the invention is that each compound may beused at low doses in a combination therapy, while producing, incombination, a substantial clinical benefit to the patient. Thecombination therapy may indeed be effective at doses where the compoundshave individually low or no effect. Accordingly, a particular advantageof the invention lies in the ability to use sub-optimal doses of eachcompound, i.e., doses which are lower than therapeutic doses usuallyprescribed, preferably ½ of therapeutic doses, more preferably ⅓, ¼, ⅕,or even more preferably 1/10 of therapeutic doses. In particularexamples, doses as low as 1/20, 1/30, 1/50, 1/100, or even lower, oftherapeutic doses are used.

In a particular embodiment such conjunction of molecular effects canfurther lead to synergistic combinations. Synergy may be assessed bymethods known well known by those skilled in the art. For instance,synergy can be characterized by using a two way ANOVA to determinewhether the interaction between each drugs is significant or not (i.e.synergy, [40]), or by calculating a combinatory index from the doseeffect curves of each of the compounds alone and of their combinations[41,42].

At such sub-therapeutic dosages, the compounds would exhibit no sideeffect, while the combination(s) according to the invention are fullyeffective in treating ALS or a related disorder.

A preferred dosage corresponds to amounts from 1% up to 50% of thoseusually prescribed for long-term maintenance treatment.

The most preferred dosage may correspond to amounts from 1% up to 10% ofthose usually prescribed for long-term maintenance treatment.

A therapeutically effective amount of cilostazol is an amount suitablefor preventing or reducing the risk of developing ALS disease andhalting or slowing the progression of ALS disease once it has becomeclinically manifest.

Preferred examples of dosages according to the invention are:

-   -   mexiletine: from about 6 to 120 mg per day, preferably less than        60 mg per day, more preferably less than 30 mg per day, even        more preferably less than 15 mg per day, such dosages being        particularly suitable for oral administration,    -   torasemide: from about 0.05 to 4 mg per day, preferably less        than 2 mg per day, more preferably less than 1 mg per day, even        more preferably less than 0.5 mg per day, such dosages being        particularly suitable for oral administration,    -   acamprosate: between 1 to 1000 mg per day, preferably less than        500 mg per day, preferably less than 400 mg per day, more        preferably less than 200 mg per day, even more preferably less        than 50 mg per day, or even less than 10 mg per day, even more        preferably from about 0.1 to 100 mg per day, furthermore        preferably between 0.5 mg and 100 mg, typically 0.8 mg per day,        2 mg per day, 20 mg per day, 40 mg per day, or 80 mg per day,        such dosages being particularly suitable for oral        administration,    -   baclofen: between 0.01 to 150 mg per day, preferably less than        100 mg per day, more preferably less than 50 mg per day, even        more preferably less than 30 mg per day, typically 12 mg per        day, 24 mg per day, 30 mg per day, such dosages being        particularly suitable for oral administration,    -   cinacalcet: from about 0.3 to 150 mg per day, preferably less        than 100 mg per day, preferably less than 50 mg per day, more        preferably less than 36 mg per day, and even more preferably        between 0.3 and 25 mg per day, such dosages being particularly        suitable for oral administration,    -   sulfisoxazole: 800 mg per day or less, preferably less than 400        mg, more preferably less than 200 mg per day, more preferably        less than 100 mg per day, even more preferably less than 20 mg        per day, such dosages being particularly suitable for oral        administration,    -   riluzole: from about 0.01 to 100 mg per day, preferably less        than 75 mg per day, more preferably less than 50 mg per day,        even more preferably less than 25 mg per day, such dosages being        particularly suitable for oral administration.

A more particular object of the invention is a tablet suitable for theadministration of any of therapy of the invention, comprising baclofen,acamprosate and riluzole. In an even more particular embodiment, saidtablet is cleavable in 2, 3 and/or 4 part as a function of the dose tobe administered at each taking.

Alternatively, where a separate administration would be considered moreproper, combinations of the invention can be provided under the form ofa unit dosage package, such unit dosage package being configured to holda first unit dosage comprising acamprosate and baclofen and a secondunit dosage comprising riluzole. In a particular embodiment unit dosagesare tablets.

It will be understood that the amount of the drug actually administeredwill be determined by a physician, in the light of the relevantcircumstances including the condition or conditions to be treated, theexact composition to be administered, the age, weight, and response ofthe individual patient, the severity of the patient's symptoms, and thechosen route of administration. Additionally, pharmacogenomic (theeffect of genotype on the pharmacokinetic, pharmacodynamic or efficacyprofile of a therapeutic) information about a particular patient mayaffect the dosage used. Therefore, the above dosage ranges are intendedto provide general guidance and support for the teachings herein, butare not intended to limit the scope of the invention.

Although the active drugs of the present invention may be administeredin divided doses, for example two or three times daily, a single dailydose of each drug in the combination is preferred, with a single dailydose of all drugs in a single pharmaceutical composition (unit dosageform) being most preferred. The term “unit dosage form” refers tophysically discrete units (such as capsules, tablets, or loaded syringecylinders) suitable as unitary dosages for human subjects, each unitcontaining a predetermined quantity of active material or materialscalculated to produce the desired therapeutic effect, in associationwith the required pharmaceutical carrier.

Administration can be one to several times daily for several days toseveral years, and may even be for the life of the patient. Chronic orat least periodically repeated long-term administration will beindicated in most cases.

In a most preferred embodiment, combinations of the invention are usedin a combination with riluzole, wherein all the drugs are administeredorally. In such protocol, riluzole is preferably administeredrepeatedly, e.g., daily, more preferably at a daily dosage of 0.01-100mg per day, even more preferably at a daily dosage of 0.1-100 mg perday, most preferably between 0.1-50 mg per day, and the other drugs atthe above indicated dosages.

The drugs may be administered simultaneously, i.e., approximately at thesame time, although not necessarily exactly at the same time or throughthe same formulation. In particular, riluzole may be formulatedseparately from the other drugs and all being ingested at approximatelythe same period of the day, to ensure they are present and can act incombination in the body. It is also possible to define a therapeuticprotocol where riluzole is administered in alternation with thecombination comprising of at least two drugs selected from acamprosate,baclofen, cinacalcet, mexiletine, sulfisoxazole and torasemide (e.g.,one day riluzole, the following day the said combination, and so on). Itshould be noted that various protocols may be adjusted or defined by thephysician, ensuring the combination therapy of the invention is mosteffective in each patient.

The following examples are given for purposes of illustration and not byway of limitation.

EXAMPLES Protective Effect of Drug Combinations in Models of ALS

Combination therapies according to the present invention are tested invitro, on rat cortical cells, in a nerve-muscle co-culture model, and invivo, in a mouse model of ALS. Protocols and results are presented inthis section.

All animal experiments were carried out according to the NationalInstitute of Health (NIH) guidelines for the care and use of laboratoryanimals, and approved by the National Animal Experiment Board.

1. Protective Effect Against Glutamate Toxicity in Primary Cultures ofNeuronal Cells.

Glutamate toxicity is involved in the pathogenesis of ALS. In this setof experiment, candidate compounds have been tested for their ability toprevent or reduce the toxic effects of glutamate on neuronal cells. Thedrugs are first tested individually, followed by assays of theircombinatorial action.

Neuronal Cell Preparation

The efficacy of drug combinations of the invention was first assessed onprimary cortical neuron cells.

Rat cortical neurons were cultured as described by Singer et al. [43].Briefly pregnant female rats of 15 days gestation were killed bycervical dislocation (Rats Wistar) and the foetuses were removed fromthe uterus. The cortex was removed and placed in ice-cold medium ofLeibovitz (L15) containing 2% of penicillin 10.000 U/mL and streptomycin10 mg/mL and 1% of bovine serum albumin (BSA). Cortices were dissociatedby trypsin for 20 min at 37° C. (0.05%). The reaction was stopped by theaddition of Dulbecco's modified Eagle's medium (DMEM) containing DNaselgrade II and 10% of foetal calf serum (FCS). Cells were thenmechanically dissociated by 3 serial passages through a 10 mL pipetteand centrifuged at 515 g for 10 min at +4° C. The supernatant wasdiscarded and the pellet of cells was re-suspended in a defined culturemedium consisting of Neurobasal supplemented with B27 (2%), L-glutamine(0.2 mM), 2% of PS solution and 10 ng/mL of brain-derived neurotrophicfactor (BDNF). Viable cells were counted in a Neubauer cytometer usingthe trypan blue exclusion test. The cells were seeded at a density of 30000 cells/well in 96 well-plates (wells were pre-coated withpoly-L-lysine (10 μg/mL)) and were cultured at +37° C. in a humidifiedair (95%)/CO2 (5%) atmosphere.

Glutamate Toxicity Assays

The neuroprotective effect of compounds was assessed by quantificationof the neurite network (neurofilament immunostaining) which specificallyreveals the glutamatergic neurons.

After 12 days of neuron culture, drugs of the candidate combinationswere solved in culture medium (+0.1% DMSO). Candidate combinations werethen pre-incubated with neurons for 1 hour before the Glutamate injury.One hour after incubation with candidate combinations, glutamate wasadded for 20 min, to a final concentration of 40 μM, in presence ofcandidate combinations, in order to avoid further drug dilutions. At theend of the incubation, medium was changed with medium with candidatecombination but without glutamate. The culture was fixed 24 hours afterglutamate injury. MK801 (dizocilpinehydrogen maleate, 77086-22-7-20 μM)was used as a positive control.

After permeabilization with saponin (Sigma), cells were blocked for 2hours with PBS containing 10% goat serum, then the cells were incubatedwith mouse monoclonal primary antibody against Neurofilament antibody(NF, Sigma). This antibody was revealed with Alexa Fluor 488 goatanti-mouse IgG.

Nuclei of cells were labeled by a fluorescent marker (Hoechst solution,Sigma), and neurite network quantified. Six wells per condition wereused to assess neuronal survival in 3 different cultures.

Results

All of the tested drug combinations give a protective effect againstglutamate toxicity for cortical neuronal cells. Results are shown inTable 2 below.

As exemplified in FIGS. 1, 3 and 4, combinations of the inventionstrongly protect neurons from glutamate toxicity under the experimentalconditions described above. It is noteworthy that an effectiveprotection is noticed using drug concentrations at which drugs usedalone have no significant or lower protective effect.

Indeed, as exemplified in FIG. 3, mexiletine-cinacalcet combinationefficiently protects neuronal cells from glutamate toxicity, whereas noprotection is afforded by the single drugs. Baclofen-acamprosate(FIG. 1) combination gives a protective effect against glutamatetoxicity for cortical neuronal cells. Combination of baclofen andacamprosate induces an improvement of more than 200% compared toacamprosate alone and more than 47% compared to baclofen used alone.Sulfisoxazole-torasemide combination (FIG. 4) induces an improvement ofabout 400% than that provided by torasemide alone at the sameconcentration, and of more than 800% than that observed whensulfisoxazole is used alone at the same concentration.

TABLE 2 Neuroprotective effect against Drug Combination glutamatetoxicity baclofen and torasemide + baclofen-acamprosate-torasemide +mexiletine and cinacalcet + sulfisoxazole and torasemide + baclofen andacamprosate + acamprosate and cinacalcet + baclofen and cinacalcet +

2. Protective Effect Against Glutamate Toxicity in Primary Cultures ofNerve-Muscle Co-Culture.

Primary Cocultures of Nerve- and Muscle Cells

Human muscle is prepared according to a previously described method fromportions of biopsy of a healthy subject [44]. Muscle cells areestablished from dissociated cells (20 000 cells per wells), plated ingelatin-coated 0.1% on 48 wells plate and grown in a proliferatingmedium consisting of mix of 75% of MEM medium with 25% of M199 mediumsupplemented with glutamine 2 mM, bovine insulin 10 μg/mL, humanrecombinant epidermal growth factor 10 ng/mL, human recombinantfibroblast growth factor basic 2 ng/mL, FCS 10% and 2% penicillin 10 000U/mL and streptomycin (10 mg/mL).

Immediately after satellite cells fusion, whole transverse slices of13-day-old rat Wistar embryos spinal cords with 4 dorsal root ganglia(DRG) attached are placed on the muscle monolayer (one explant per well,in center area). DRG are necessary to achieve a good ratio ofinnervations. Innervated cultures are maintained in a mixed medium (75%of MEM with 25% of M199), supplemented with glutamine 2 mM, 5% FCS,bovine insulin 5 μg/mL, and 2% penicillin 10 000 U/mL and streptomycin(10 mg/mL).

After 24 hours of co-culture, neurites are observed growing out of thespinal cord explants. They make contacts with myotubes and induce thefirst contractions after about 8 days. Quickly thereafter, innervatedmuscle fibres located in proximity to the spinal cord explants, arevirtually continuously contracting. Innervated fibres aremorphologically and spatially distinct from the non-innervated ones andcould easily be distinguished from them.

Glutamate Injury

On day 27, co-cultures are incubated with candidate compounds,combination thereof or riluzole one hour before glutamate intoxication(60 μM) for 20 min. Then, co-cultures are washed and candidatecompounds, combination thereof, and/or riluzole are added for anadditional 48 hours. After this incubation time, unfixed co-cultures areincubated with α-bungarotoxin coupled with Alexa 488 at concentration500 nmol/L for 15 min at room temperature. Then, co-cultures fixed byparaformaldehyde for 20 min at room temperature. After permeabilizationwith 0.1% of saponin, co-cultures are incubated with NF (dilution1/400).

These antibodies are detected with Alexa Fluor 568 goat anti-mouse IgG(Molecular probe, 1/400 dilution), nuclei of neurons are labeled by afluorescent marker (Hoechst solution, 1 μg/mL in the same solution).

Endpoints are (1) total neurite length, (2) number of motor units, (3)total motor unit area, which are indicative of motor neuron survival andfunctionality.

For each condition, 2×10 pictures per well are taken using InCellAnalyzer™ 1000 (GE Healthcare) with 20× magnification. All the imagesare taken in the same conditions.

Glutamate Injury with Riluzole Pre-Treatment

On day 23 (i.e. 4 days of pre-treatment), co-cultures are incubated withriluzole. After 4 days (i.e. on day 27), drug combinations are added onehour before glutamate addition and then glutamate (60 μM) is added for20 min. Cocultures are then treated for immunofluorescence analysis asstated above.

In another set of experiments, the same protocol than above is performedexcept that riluzole is removed from cultures when said drugcombinations are added (i.e. after the 4 days riluzole pre-treatment)(“switch” experiments).

Results

A significant protection is noticed for all the three endpoints whendrugs are used in combination, at concentrations where, when used alone,no effect is noticed. Tested drug combinations are listed in Table 3 andexemplified in FIG. 2. This unexpected synergistic effect allows usingdrugs at doses so low that potential side effects should be overcome.

TABLE 3 Protective effect against glutamate intoxication DrugCombination in muscle/nerve co-cultures baclofen and cinacalcet +cinacalcet and acamprosate + baclofen-torasemide +baclofen-acamprosate-torasemide + mexiletine and cinacalcet +sulfisoxazole and torasemide + baclofen and acamprosate +

Drug combinations of the invention enhance the protective effect of theriluzole toward glutamate toxicity, in the in vitro model, for the threeendpoints, with the observation of synergy between the two treatments(Table 4, FIG. 7).

TABLE 4 Enhancement of the protective effect of riluzole againstglutamate intoxication in Drug Combination the muscle/nerve co-culturesbaclofen-cinacalcet-riluzole + cinacalcet-acamprosate-riluzole +baclofen-torasemide-riluzole +baclofen-acamprosate-torasemide-riluzole +mexiletine-cinacalcet-riluzole + sulfisoxazole-torasemide-riluzole +baclofen-acamprosate-riluzole +

The inventors have found that combinations of the invention potentiateriluzole protective effect in the muscle/nerve co-culture model. Indeed,baclofen-acamprosate adjunction to the riluzole treated glutamateintoxicated cells results in an improvement of protection afforded tothe cells. Moreover, as shown in the FIG. 5, the inventors have beenable to identify drug concentrations at which this enhancing effect isparticularly important (acamprosate 0.14 nM and baclofen 36 nM). Thesame is observed for the three endpoints, with other concentrations ofriluzole (0.04 and 0.5 μM) and other concentrations of acamprosate andbaclofen (0.14 nM and 32 nM, respectively).

As exemplified in FIG. 6, drug combinations of the invention actsynergistically with riluzole to protect neuromuscular junctions againstglutamate toxicity. Noteworthy, used at a dose barely efficient (36 nMand 0.14 nM of baclofen and acamprosate respectively), the addition ofbaclofen-acamprosate mix in the culture medium of riluzole treated cellsresults in almost the doubling (or even more) of the protective effectof riluzole against glutamate injuries.

Switching from riluzole, after the riluzole pre-treatment of 96 hours,toward the combination of acamprosate with baclofen improves the benefitprovided by either the riluzole or the baclofen-acamprosate singletreatments in the three endpoints of ALS in vitro co-culture model (FIG.8).

Thus, compositions of the invention are also particularly efficient asadjunctive therapy for other ALS treatments (more particularly,riluzole) or ALS candidate treatments.

3. Combinations Therapies are Efficient in ALS Mouse Model.

Transgenic heterozygous mice B6SJL-Tg(SOD1-G93A)1Gur/J mice and WT mice(strain 1012, JAX) have been chosen to mimic ALS in this set ofexperiments. Diseased mice express the SOD1-G93A transgene, designedwith a mutant human SOD1 gene (a single amino acid substitution ofglycine to alanine at codon 93) driven by its endogenous human SOD1promoter.

Animals are housed at a standard temperature (22° C.±1° C.) and in alight-controlled environment (lights on from 7 am to 8 pm) with adlibitum access to food and water. Starting at the age of 100 days allG93A SOD1 mice receive wet powdered food (Standard Lab Diet mixed withwater to form a paste) and nutritional gel placed in the cage. Inaddition, water spouts are fitted with extensions to allow mice toeasily access from floor level.

Drug Administration

Mice dosed with candidate drug treatment diluted in vehicle from 60^(th)day after birth till the mice reach 150 days of age. Diluted solutionsof drug candidates are prepared with water at room temperature justbefore the beginning of the administration. Both riluzole and drugcombinations are administrated per os. Cyclodextrin is used as vehicleat the final concentration of 5%, diluted in water at room temperaturefrom stock solution (cyclodextrin 20%). Treatment with drug combinations(10 mL/kg) and vehicle starts at the age of 60 days and continues untilthe mice reach 150 days of age. Drug combinations were administered peros bis in die (bid) between 8-11 am and 4-7 pm.

Experimental Set Up of Mice

In setting up groups for study (i.e. test or control article treated),transgenic mice are randomized into groups so that whole litters of micedo not end up in a single testing group, thereby avoiding ‘littereffects’ on the overall results. In addition, the groups are equallybalanced for the male/female ratio.

Female mice are housed in groups of 5 maximum and male mice are singlehoused. Mice are allowed to acclimate to the experimental room for atleast one hour prior to the beginning of any experiment. Mice aretransported from the colony room to experimental rooms in their homecages.

Body Weight

Weight loss has proven to correlate well with disease development and iseasily scored co-jointly with disease stages. The mice will be weighedonce-a-week on the same day each week (Mon) at the age of 60-91 days andthree times a week (Mon-Wed-Fri) after they reach the age of 91 days (13weeks).

Clinical Scoring

The original article [45] describing the generation of the SOD1-G93Amice reports an early onset of the disease (ca. 100 days) and a rapiddecline with the affected mice reaching the end stage on average within40 days (typical survival 130-150 days). Hence, the mice are carefullyexamined using clinical scoring as described below once a week (Mon)until age of 91 days and three times a week (Mon-Wed-Fri) after theyreach the age of 91 days.

The earliest clinical signs are tremors and shaking of their limbs whenthe mice are suspended briefly in the air by their tails. The clinicalscoring system is on a scale of 1 to 5; with 1 as the endpoint foreuthanasia, and 5 as healthy with little or no signs of onset ofdisease. Animals are scored by lifting them gently by the base of theirtails and observing them for tremors, stiffness and their ability toextend their limbs.

Scoring System:

5=healthy4-5=mostly healthy, minor tremors, very active, extension of all limbs4=visible minor tremors, extension of all limbs, very active3-4=tremors, with some minor stiffness, very active3=tremors, stiffness of limbs, maybe some minor paralysis, active2-3=tremors, partial paralysis, stiffness, extension of limbs islabored, active2=paralysis, somewhat active1-2=paralysis of hind limbs, no extension of hind limbs, euthanasia maybe performed dependent upon the activity of the animal and its abilityto right itself within 30 sec1=endpoint, animal unable to right itself.The onset of disease is recorded when the score reaches a disease stageof 4.

Behavioral Testing

All behavioral tests are stopped at the age of 20 weeks whenapproximately 70% of the vehicle group TG mice are lost. After this agethe remaining mice are too fragile for motoric testing and are onlysubjected to body weight measurement, disease stage and survivalscoring.

Open Field Test

Open field test measurements are performed before the dosing is started(baseline) and around day 90 (13^(th) age week) and day 110 (16^(th) ageweek). Mice born within 2-4 days are pooled for open field testing.Activity chambers (Med Associates Inc., St Albans, Vt.; 27×27×20.3 cm)are equipped with IR beams. Mice are placed in the center of the chamberand their behavior is recorded for 10 min. Distance moved, number ofvertical rearings and average velocity are recorded.

Rotarod

Rotarod test is performed before the dosing is started (baseline) andaround day 90 (13^(th) age week) and day 110 (16^(th) age week): Miceborn within 2-4 days are pooled for open field testing. One day sessionincludes a training trial of 5 min at 4 revolutions per minute (RPM) onthe rotarod apparatus (AccuScan Instruments, Columbus, USA). One hourlater, the animals are tested for 3 consecutive accelerating trials of 6min with the speed changing from 0 to 40 RPM over 360 sec and aninter-trial interval at least 30 min. The latency to fall from the rodis recorded. Mice remaining on the rod for more than 360 sec are removedand their time scored as 360 sec.

Results

Combinations therapies are efficient in ALS in vivo model.

An improvement of the disease is observed for the diseased animalstreated with the drug combinations of the invention. Notably, drugcombinations of the invention efficiently improve clinical score ofthese animals during the different stages of the disease (Table 5) andalso performances in the above behavioural tests (Table 6).

TABLE 5 Drug Combination Improvement of Global clinical score baclofenand cinacalcet + cinacalcet and acamprosate + baclofen-torasemide +baclofen-acamprosate-torasemide + mexiletine and cinacalcet +sulfisoxazole and torasemide + baclofen and acamprosate +

TABLE 6 Behavioral testing Improvement of performances Drug Combinationrotarod test open field test baclofen and cinacalcet + + cinacalcet andacamprosate + + baclofen-torasemide + +baclofen-acamprosate-torasemide + + mexiletine and cinacalcet + +sulfisoxazole and torasemide + + baclofen and acamprosate + +

The compositions of the invention are also efficient in improvingclinical score and course of the disease in riluzole treated animals(Table 7).

TABLE 7 Improvement of Global clinical score in Drug Combinationriluzole treated animals baclofen and cinacalcet + cinacalcet andacamprosate + baclofen-torasemide + baclofen-acamprosate-torasemide +mexiletine and cinacalcet + sulfisoxazole and torasemide + baclofen andacamprosate +

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We claim:
 1. A method of treating amyotrophic lateral sclerosis (ALS) ora related motor neuron disorder in a subject suffering thereof, themethod comprising administering to the subject an effective amount of acomposition comprising mexiletine and cinacalcet, or salts, or prodrugs,or derivatives, or sustained-release formulations thereof.
 2. The methodof claim 1, for reducing motor neuron degeneration in said subject. 3.The method of claim 1, comprising administering mexiletine, cinacalcetand riluzole, or salts, or prodrugs, or derivatives, orsustained-release formulations thereof.
 4. The method of claim 1,wherein the compounds are administered with a pharmaceuticallyacceptable carrier or excipient.
 5. The method of claim 1, wherein thecompounds are administered repeatedly to the subject.
 6. The method ofclaim 1, wherein the compounds are formulated or administered together,separately or sequentially.
 7. The method of claim 6, wherein thecompounds are formulated together.
 8. The method of claim 3, whereinmexiletine and cinacalcet, or the salts, or prodrugs, or derivatives, orsustained-release formulations thereof, are administered alternatelywith riluzole, or a salt, prodrug, or derivative, or sustained-releaseformulation thereof.
 9. The method of claim 1, wherein mexiletine isadministered in a dosage from about 6 to 120 mg per day.
 10. The methodof claim 1, wherein cinacalcet is administered in a dosage from about0.3 to 150 mg per day.
 11. The method of claim 3, wherein riluzole isadministered in a dosage from about 0.01 to 50 mg per day.
 12. Themethod of claim 1, wherein said compounds are administered orally. 13.The method of claim 3, wherein said compounds are administered orally.14. The method of claim 1, wherein said related motor neuron disorder isa disorder selected from the group consisting of primary lateralsclerosis, progressive muscular atrophy, pseudobulbar palsy andprogressive bulbar palsy, and fronto temporal dementia.
 15. A method forprotecting neuromuscular junctions against glutamate toxicity in asubject in need thereof, comprising administering to the subject acomposition comprising mexiletine, cinacalcet and riluzole.
 16. Apharmaceutical composition comprising mexiletine, cinacalcet andriluzole, or salts, or prodrugs, or derivatives, or sustained-releaseformulations thereof.